The invention pertains to compounds that inhibit poly(ADP-ribose) polymerases, thereby retarding the repair of damage to DNA strands, and to methods of preparing such compounds. The invention also relates the use of such compounds in pharmaceutical compositions and therapeutic treatments useful for potentiation of anti-cancer therapies and inhibition of neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases.
Poly(ADP-ribose) polymerases (PARPs), nuclear enzymes found in almost all eukaryotic cells, catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to nuclear acceptor proteins, and are responsible for the formation of protein-bound linear and branched homo-ADP-ribose polymers. Activation of PARP and resultant formation of poly(ADP-ribose) can be induced by DNA strand breaks after exposure to chemotherapy, ionizing radiation, oxygen free radicals, or nitric oxide (NO).
Because this cellular ADP-ribose transfer process is associated with the repair of DNA strand breakage in response to DNA damage caused by radiotherapy or chemotherapy, it can contribute to the resistance that often develops to various types of cancer therapies. Consequently, inhibition of PARP may retard intracellular DNA repair and enhance the antitumor effects of cancer therapy. Indeed, in vitro and in vivo data show that many PARP inhibitors potentiate the effects of ionizing radiation or cytotoxic drugs such as DNA methylating agents. Therefore, inhibitors of the PARP enzyme are useful as cancer chemotherapeutics.
In addition, it has been shown that inhibition of PARP promotes resistance to brain injury after stroke (Endres et al., xe2x80x9cIschemic Brain Injury is Mediated by the Activation of Poly(ADP-Ribose)Polymerase,xe2x80x9d J. Cerebral Blood Flow Metab. 17:1143-1151 (1997); Zhang, xe2x80x9cPARP Inhibition Results in Substantial Neuroprotection in Cerebral Ischemia,xe2x80x9d Cambridge Healthtech Institute""s Conference on Acute Neuronal Injury: New Therapeutic Opportunities, Sep. 18-24, 1998, Las Vegas, Nev.). The activation of PARP by DNA damage is believed to play a role in the cell death consequent to stroke, head trauma, and neurodegenerative diseases. DNA is damaged by excessive amounts of NO produced when the NO synthase enzyme is activated as a result of a series of events initiated by the release of the neurotransmitter glutamate from depolarized nerve terminals (Cosi et al., xe2x80x9cPoly(ADP-Ribose) Polymerase Revisited: A New Role for an Old Enzyme: PARP Involvement in Neurodegeneration and PARP Inhibitors as Possible Neuroprotective Agents,xe2x80x9d Ann. N.Y. Acad. Sci., 366-379). Cell death is believed to occur as a result of energy depletion as NAD+ is consumed by the enzyme-catalyzed PARP reaction. Therefore, inhibitors of the PARP enzyme are useful inhibitors of neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases.
Further, inhibition of PARP should be a useful approach for treatment of conditions or diseases associated with cellular senescence, such as skin aging, through the role of PARP in the signaling of DNA damage. See, e.g., U.S. Pat. No. 5,589,483, which describes a method to extend the lifespan and proliferative capacity of cells comprising administering a therapeutically effective amount of a PARP inhibitor to the cells under conditions such that PARP activity is inhibited. Hence, inhibitors of the PARP enzyme are useful therapeutics for skin aging.
In yet a further application, PARP inhibition is being explored at the clinical level to prevent development of insulin-dependent diabetes mellitus in susceptible individuals (Saldeen et al., xe2x80x9cNicotinamide-induced apoptosis in insulin producing cells in associated with cleavage of poly(ADP-ribose) polymerase,xe2x80x9d Mol. Cellular Endocrinol. (1998), 139:99-107). PARP inhibitors should therefore be useful as diabetes-prevention therapeutics.
PARP inhibition is also an approach for treating inflammatory conditions such as arthritis (Szabo et al., xe2x80x9cProtective effect of an inhibitor of poly(ADP-ribose) synthetase in collagen-induced arthritis,xe2x80x9d Portland Press Proc. (1998), 15:280-281; Szabo, xe2x80x9cRole of Poly(ADP-ribose) Synthetase in Inflammation,xe2x80x9d Eur. J. Biochem. (1998), 350(1):1-19; Szabo et al., xe2x80x9cProtection Against Peroxynitrite-induced Fibroblast Injury and Arthritis Development by Inhibition of Poly(ADP-ribose) Synthetase,xe2x80x9d Proc. Natl. Acad. Sci. USA (1998), 95(7):3867-72). PARP inhibitors are therefore useful as therapeutics for inflammatory conditions.
Inhibition of PARP has usefulness for protection against myocardial ischemia and reperfusion injury (Zingarelli et al., xe2x80x9cProtection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase,xe2x80x9d Cardiovascular Research (1997), 36:205-215). Therefore, PARP inhibitors are useful in therapy of cardiovascular diseases.
The PARP family of enzymes is extensive. It has recently been shown that tankrases, which bind to the telomeric protein TRF-1, a negative regulator of telomere length maintenance, have a catalytic domain that is strikingly homologous to PARP and have been shown to have PARP activity in vitro. It has been proposed that telomere function in human cells is regulated by poly(ADP-ribosyl)ation. PARP inhibitors have utility as tools to study this function. Further, as a consequence of regulation of telomerase activity by tankyrase, PARP inhibitors should have utility as agents for regulation of cell life-span, e.g., for use in cancer therapy to shorten the life-span of immortal tumor cells, or as anti-aging therapeutics, since telomere length is believed to be associated with cell senescence.
Competitive inhibitors of PARP are known. For example, Banasik et al. (xe2x80x9cSpecific Inhibitors of Poly(ADP-Ribose) Synthetase and Mono(ADP-Ribosyl)transferase,xe2x80x9d J. Biol. Chem. (1992) 267: 1569-1575) examined the PARP-inhibiting activity of 132 compounds, the most potent of which were 4-amino-1,8-naphthalimide, 6(5H)-phenanthridone, 2-nitro-6(5H)-phenanthridone, and 1,5-dihydroxyisoquinoline. Griffin et al. reported the PARP-inhibiting activity for a series of benzamide compounds (U.S. Pat. No. 5,756,510; see also xe2x80x9cNovel Potent Inhibitors of the DNA Repair Enzyme poly (ADP-ribose)polymerase (PARP),xe2x80x9d Anti-Cancer Drug Design (1995), 10:507-514) and quinalozinone compounds (International Publication No. WO 98/33802). Suto et al. reported PARP inhibition by a series of dihydroisoquinoline compounds (xe2x80x9cDihydroisoquinolines: The Design and Synthesis of a New Series of Potent Inhibitors of Poly(ADP-ribose) Polymerase,xe2x80x9d Anti-Cancer Drug Design (1991), 7:107-117). Griffin et al. have reported other PARP inhibitors of the quinazoline class (xe2x80x9cResistance-Modifying Agents. 5. Synthesis and Biological Properties of Quinazoline Inhibitors of the DNA Repair Enzyme Poly(ADP-ribose) Polymerase (PARP),xe2x80x9d J. Med. Chem., ASAP Article 10.1021/jm980273t S0022-2623(98)00273-8; Web Release Date: Dec. 1, 1998).
Nonetheless, there is still a need for small-molecule compounds that are potent PARP inhibitors, especially those that have physical and chemical properties desirable for pharmaceutical applications.
The present invention is directed to compounds that function as potent poly(ADP-ribosyl)transferase (PARP) inhibitors and are useful as therapeutics, especially in treatment of cancers and the amelioration of the effects of stroke, head trauma, and neurodegenerative disease. As cancer therapeutics, the compounds of the invention may be used in combination with DNA-damaging cytotoxic agents, for example, topotecan, irinotecan, or temozolomide, and/or radiation.
In particular, the present invention is directed to compounds of the general formula (I): 
wherein:
R1 is:
H;
halogen;
cyano;
an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group (e.g., unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and amino, alkoxy, alkyl, and aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, carboxy, and optionally substituted amino and ether groups (such as O-aryl)); or
xe2x80x94C(O)xe2x80x94R10, where R10 is: H; an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group (e.g., unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and alkyl and aryl groups unsubstituted or substituted with one or more substituents selected from halo, hydroxy, nitro, and amino); or OR100 or NR100R110, where R100 and R110 are each independently H or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group (e.g., unsubstituted or substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and alkyl and aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and optionally substituted amino groups);
R2 is H or alkyl;
R3 is H or alkyl;
R4 is H, halogen or alkyl;
X is O or S;
Y is (CR5R6)(CR7R8)n or Nxe2x95x90C(R5), where:
n is 0 or 1;
R5 and R6 are each independently H or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group (e.g., unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and lower alkyl, lower alkoxy, or aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and amino); and
R7 and R8 are each independently H or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group (e.g., unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and lower alkyl, lower alkoxy, and aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and amino);
where when R1, R4, R5, R6, and R7 are each H, R8 is not unsubstituted phenyl.
The invention is also directed to pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates of such compounds. Preferred compounds of the formula (I) include those where R2 and R3 are each independently selected from H and methyl.
In a preferred embodiment, the inventive compounds include those of generic formula (II): 
wherein:
p is 1 or 2;
R11 is H or alkyl;
R12 is halogen or an optionally substituted aryl, alkyl, alkenyl, alkynyl or acyl group xe2x80x94C(O)xe2x80x94R10 as defined above;
R13 is H or alkyl; and
R14 is H or halogen;
as well as pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates of such compounds.
In preferred compounds of the formula (II), R11 and R13 are each independently selected from H and methyl. More preferably, the invention is directed to compounds of formula (II) where R11 and R13 are each H, and R12 is optionally substituted aryl, and to pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates of such compounds. In another preferred embodiment of compounds of formula (II), R11 and R13 are each H, and R12 is halogen or optionally substituted aryl.
In another preferred embodiment, the inventive compounds include those of generic formula (III) below, as well as pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates thereof. 
wherein:
R15 is H, halogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and alkyl and aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and amino;
R16 is H; halogen; cyano; or an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, amino, and alkyl and aryl groups unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, nitro, and amino;
R17 is H or alkyl; and
R18 is H, halogen, or alkyl;
where R15, R16, R17 and R18 are not all H.
In preferred compounds of the formula (III), R15 is substituted phenyl or (CH2)qaryl, where q is 1 or 2.
In other preferred compounds of the formula (III), R16 is substituted or unsubstituted aryl.
The present invention is also directed to a method of inhibiting PARP enzyme activity, comprising contacting the enzyme with an effective amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof. The compounds of the invention are potent PARP inhibitors and preferably have a PARP-inhibiting activity corresponding to a Ki of 100 xcexcM or less in the PARP enzyme inhibition assay.
The present invention is further directed to a method of potentiating the cytotoxicity of a cytotoxic drug or ionizing radiation, comprising contacting cells with an effective amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof, in combination with a cytotoxic drug or ionizing radiation. The compounds of the invention preferably have a cytotoxicity potentiation activity corresponding to a PF50 of at least 1 in the cytotoxicity potentiation assay.
The present invention is also directed to pharmaceutical compositions comprising an effective PARP-inhibiting amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof, together with a pharmaceutically acceptable carrier therefor.
The invention also provides therapeutic interventions appropriate in disease or injury states where PARP activity is deleterious to a patient, the therapeutic methods comprising inhibiting PARP enzyme activity in the relevant tissue of the patient by administering a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof. In one such therapeutic intervention method provided by the present invention, the effectiveness of a cytotoxic drug or radiotherapy administered to a mammal in the course of therapeutic treatment is improved by administering to the patient, e.g., a mammal in need of treatment, an effective PARP-inhibiting amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof, in conjunction with the administration of the cytotoxic drug or radiotherapy.
Another therapeutic intervention method provided by the present invention is for delaying the onset of cell senescence associated with skin aging in a human, comprising administering to fibroblast cells in the human an effective PARP-inhibiting amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof.
Yet another therapeutic intervention method provided by the present invention is a method for reducing the neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases in a mammal by administering an effective amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof, to the mammal.
The compounds of the present invention provide a therapeutic approach to treatment of inflammatory conditions, comprising administering an effective amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof, to a patient in need of treatment.
Yet a further therapeutic intervention method provided by the present invention is a cardiovascular therapeutic method for protecting against myocardial ischemia and reperfusion injury in a mammal, comprising administering to the mammal an effective amount of a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof.
The present invention is further directed to methods of synthesizing the tricyclic compounds of formula (I), wherein a 4-carboalkoxy indole (IV) is converted to an intermediate 3-substituted-4-carboalkoxy indole, thereby incorporating the intended ring carbon atoms, terminally substituted with one nitrogen atom, usually in the form of a nitro group. Additional functional groups, such as formyl or acyl, may be incorporated at the 3-position in this step. The nitro group is reduced to an amine and cyclized upon the 4-carboalkoxy group in an amide-forming reaction to yield the tricyclic heterocycle. The synthetic methods may further comprise derivatization at N-1 and C-2. The 3-formyl or 3-acyl intermediates can be converted to nitrogen-containing intermediates or to tricyclic indoles with Nxe2x80x94N bonds, such as the compounds of formula (III). 
PARP-Inhibiting Agents
In accordance with a convention used in the art, the symbol 
is used in structural formulas herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure. in accordance with another convention, in some structural formulae herein the carbon atoms and their bound hydrogen atoms are not explicitly depicted, e.g., 
represents a methyl group, 
represents an ethyl group, 
represents a cyclopentyl group, etc.
As used herein, the term xe2x80x9calkylxe2x80x9d means a branched- or straight-chained (linear) paraffinic hydrocarbon group (saturated aliphatic group) having from 1 to 10 carbon atoms in its chain, which may be generally represented by the formula CkH2k+1, where k is an integer of from 1 to 10. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, n-pentyl, isopentyl, neopentyl, and hexyl, and the simple aliphatic isomers thereof. A xe2x80x9clower alkylxe2x80x9d is intended to mean an alkyl group having from 1 to 4 carbon atoms in its chain.
The term xe2x80x9calkenylxe2x80x9d means a branched- or straight-chained olefinic hydrocarbon group (unsaturated aliphatic group having one or more double bonds) containing 2 to 10 carbons in its chain. Exemplary alkenyls include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, isobutenyl, and the various isomeric pentenyls and hexenyls (including both cis and trans isomers).
The term xe2x80x9calkynylxe2x80x9d means a branched or straight-chained hydrocarbon group having one or more carbon-carbon triple bonds, and having from 2 to 10 carbon atoms in its chain. Exemplary alkynyls include ethynyl, propynyl, 1-butynyl, 2-butynyl, and 1-methyl-2-butynyl.
The term xe2x80x9ccarbocyclexe2x80x9d refers to a saturated, partially saturated, unsaturated, or aromatic, monocyclic or fused or non-fused polycyclic, ring structure having only carbon ring atoms (no heteroatoms, i.e., non-carbon ring atoms). Exemplary carbocycles include cycloalkyl, aryl, and cycloalkyl-aryl groups.
The term xe2x80x9cheterocyclexe2x80x9d refers to a saturated, partially saturated, unsaturated, or aromatic, monocyclic or fused or non-fused polycyclic, ring structure having one or more heteroatoms selected from N, O, and S. Exemplary heterocycles include heterocycloalkyl, heteroaryl, and heterocycloalkyl-heteroaryl groups.
A xe2x80x9ccycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent, monocyclic or fused polycyclic, ring structure having a total of from 3 to 18 carbon ring atoms (but no heteroatoms). Exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, phenanthrenyl, and like groups.
A xe2x80x9cheterocycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent, monocyclic or fused polycyclic, ring structure having a total of from 3 to 18 ring atoms, including 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heterocycloalkyl groups include pyrrolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, aziridinyl, and like groups.
The term xe2x80x9carylxe2x80x9d means an aromatic monocyclic or fused polycyclic ring structure having a total of from 4 to 18, preferably 6 to 18, ring carbon atoms (no heteroatoms). Exemplary aryl groups include phenyl, naphthyl, anthracenyl, and the like.
A xe2x80x9cheteroaryl groupxe2x80x9d is intended to mean an aromatic monovalent, monocyclic or fused polycyclic, ring structure having from 4 to 18, preferably 5 to 18, ring atoms, including from 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl groups include pyrrolyl, thienyl, oxazolyl, pyrazolyl, thiazolyl, furyl, pyridinyl, pyrazinyl, triazolyl, tetrazolyl, indolyl, quinolinyl, quinoxalinyl, and the like.
The term xe2x80x9coptionally substitutedxe2x80x9d is intended to indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents. Unless indicated otherwise (e.g., by indicating that a specified group is unsubstituted), the various groups defined above may be generally unsubstituted or substituted (i.e., they are optionally substituted) with one or more suitable substituents.
The term xe2x80x9csubstituentxe2x80x9d or xe2x80x9csuitable substituentxe2x80x9d is intended to mean any substituent for a group that may be recognized or readily selected by the artisan, such as through routine testing, as being pharmaceutically suitable. Illustrative examples of suitable substituents include hydroxy, halogen (F, Cl, I, or Br), oxo, alkyl, acyl, sulfonyl, mercapto, nitro, alkylthio, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxy, amino (primary, secondary, or tertiary), carbamoyl, aryloxy, heteroaryloxy, arylthio, heteroarylthio, and the like (e.g., as illustrated by the exemplary compounds described herein). Suitable substituents are seen from the exemplary compounds that follow.
Preferred optional substituents for alkyl and aryl groups in the compounds of the invention include halogens and aryl groups. Especially preferred for substituted alkyl groups are perfluoro-substituted alkyls. Especially preferred optional substituents for aryl moieties include halogen, lower alkyl, xe2x80x94OH, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, O-lower alkyl, aryl, xe2x80x94O-aryl, aryl-lower alkyl, xe2x80x94CO2CH3, xe2x80x94CONH2, xe2x80x94OCH2CONH2, xe2x80x94NH2, xe2x80x94SO2NH2, xe2x80x94OCHF2, xe2x80x94CF3, xe2x80x94OCF3, and the like. Aryl moieties may also be optionally substituted by two substituents forming a bridge, for example xe2x80x94Oxe2x80x94(CH2)zxe2x80x94Oxe2x80x94, where z is an integer of 1, 2, or 3.
A xe2x80x9cprodrugxe2x80x9d is intended to mean a compound that is converted under physiological conditions or by solvolysis, or metabolically, to a specified compound that is pharmaceutically active.
An xe2x80x9cactive metabolitexe2x80x9d is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound.
A xe2x80x9csolvatexe2x80x9d is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
A xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is intended to mean a salt that retains the biological effectiveness of the free-acid or base form of the specified compound and that is pharmaceutically suitable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, xcex3-hydroxybutyrates, glycollates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If an inventive compound is a base, a desired salt may be prepared by any suitable method known in the art, including treatment of the free base with: an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid such as glucuronic acid or galacturonic acid; alpha-hydroxy acid such as citric acid or tartaric acid; amino acid such as aspartic acid or glutamic acid; aromatic acid such as benzoic acid or cinnamic acid; sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid; or the like.
If an inventive compound is an acid, a desired salt may be prepared by any suitable method known in the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali metal or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include: organic salts derived from amino acids such as glycine and arginine; ammonia; primary, secondary, and tertiary amines; and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
In the case of compounds, salts, or solvates that are solids, it is understood by those skilled in the art that the inventive compounds, salts, and solvates may exist in different crystalline or polymorph forms, all of which are intended to be within the scope of the present invention and specified formulas.
In some cases, the inventive compounds will have chiral centers. When chiral centers are present, the inventive compounds may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the broad scope of the generic structural formulae (unless otherwise indicated). Preferably, however, the inventive compounds are used in essentially optically pure form (as generally understood by those skilled in the art, an optically pure compound is one that is enantiomerically pure). Preferably, the compounds of the invention are at least 90% of the desired single isomer (80% enantiomeric excess), more preferably at least 95% (90% e.e.), even more preferably at least 97.5% (95% e.e.), and most preferably at least 99% (98% e.e.).
In some cases, compounds can occur in tautomeric forms. In such cases, it is intended that both tautomers are encompassed by the structural formulae.
The present invention is directed to the following PARP-inhibiting agents: compounds of the formula 
wherein R1, R2, R3, R4, X, and Y are as defined above; and pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates thereof. In preferred embodiments, the PARP-inhibiting agents are compounds of the formula (I) where R2 and R3 are each independently H or methyl, and pharmaceutically acceptable salts, prodrugs, active metabolites, and solvates thereof.
More preferably, the agents are compounds of formula (II) or (III): 
wherein the variables are as defined above, or pharmaceutically acceptable salts, prodrugs, active metabolites, or solvates thereof. In preferred embodiments for formula (II) and (III), R11, R13, and R17 are each independently H or methyl.
In a preferred embodiment, the inventive agents are compounds of formula (II) and pharmaceutically acceptable salts, prodrugs, active metabolites and solvates, where R11 and R13 are each H, and R12 is an optionally substituted aryl group. In another preferred embodiment, the inventive agents are compounds of formula (III) and pharmaceutically acceptable salts, prodrugs, active metabolites and solvates, where R17 is H or methyl and R15 is optionally substituted aryl or alkyl.
In other preferred embodiments, R16 is substituted or unsubstituted aryl and R15 is hydrogen.
In other preferred embodiments, R16 is H, and R15 is substituted or unsubstituted aryl or alkyl.
Preferred compounds of the invention include: 
Pharmaceutical Methods and Compositions
The invention is also directed to a method of inhibiting PARP enzyme activity, comprising contacting the enzyme with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof. For example, PARP activity may be inhibited in mammalian tissue by administering a compound of formula (I) or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof. In addition to the compounds specified above, the following known compounds have been found to be useful for inhibiting PARP enzyme activity: 
xe2x80x9cTreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d is intended to mean mitigating or alleviating an injury or a disease condition in a mammal, such as a human, that is mediated by the inhibition of PARP activity, such as by potentiation of anti-cancer therapies or inhibition of neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases. Types of treatment include: (a) as a prophylactic use in a mammal, particularly when the mammal is found to be predisposed to having the disease condition but not yet diagnosed as having it; (b) inhibition of the disease condition; and/or (c) alleviation, in whole or in part, of the disease condition.
One treatment method involves improving the effectiveness of a cytotoxic drug or radiotherapy administered to a mammal in the course of therapeutic treatment, comprising administering to the mammal an effective amount of an agent (compound, pharmaceutically acceptable salt, prodrug, active metabolite, or solvate) in conjunction with administration of the cytotoxic drug (e.g., topotecan or irinotecan) or radiotherapy. The PARP-inhibiting agents may also be advantageously used in a method for reducing neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases in a mammal by administering a therapeutically effective amount of an inventive agent to the mammal. The PARP-inhibiting agents of the invention may also be used in a method for delaying the onset of cell senescence associated with skin aging in a human, comprising administering to fibroblast cells in the human an effective PARP-inhibiting amount of an agent. Further, the agents may also be used in a method for helping prevent the development of insulin-dependent diabetes mellitus in a susceptible individual, comprising administering a therapeutically effective amount of an agent. Additionally, the agents may also be employed in a method for treating an inflammatory condition in a mammal, comprising administering a therapeutically effective amount of an agent to the mammal. Moreover, the agents may also be used in a method for treating cardiovascular disease in a mammal, comprising administering to the mammal a therapeutically effective amount of a PARP-inhibiting agent. As knowledge regarding the therapeutic roles of PARP inhibitors progresses in the art, other utilities of the PARP-inhibiting agents of the invention will become apparent.
The activity of the inventive compounds as inhibitors of PARP activity may be measured by any of the suitable methods known or available in the art, including by in vivo and in vitro assays. An example of a suitable assay for activity measurements is the PARP enzyme inhibition assay described herein.
Administration of the compounds of the formula (I) and their pharmaceutically acceptable prodrugs, salts, active metabolites, and solvates may be performed according to any of the accepted modes of administration available in the art. Illustrative examples of suitable modes of administration include oral, nasal, parenteral, topical, transdermal, and rectal delivery. Oral and intravenous delivery are preferred.
An inventive compound of formula (I) or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof may be administered as a pharmaceutical composition in any pharmaceutical form recognizable to the skilled artisan as being suitable. Suitable pharmaceutical forms include solid, semisolid, liquid, or lyophilized formulations, such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols. Pharmaceutical compositions of the invention may also include suitable excipients, diluents, vehicles, and carriers, as well as other pharmaceutically active agents (including other PARP-inhibiting agents), depending upon the intended use.
Acceptable methods of preparing suitable pharmaceutical forms of the pharmaceutical compositions are known or may be routinely determined by those skilled in the art. For example, pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.
Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulphate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid carriers include syrup, peanut oil, olive oil, saline solution, and water. The carrier or diluent may include a suitable prolonged-release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or a nonaqueous or aqueous liquid suspension.
A dose of the pharmaceutical composition contains at least a therapeutically effective amount of a PARP-inhibiting agent (i.e., a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof), and preferably contains one or more pharmaceutical dosage units. The selected dose may be administered to a mammal, for example, a human patient, in need of treatment of a condition mediated by inhibition of PARP activity, by any known or suitable method of administering the dose, including: topically, for example, as an ointment or cream; orally; rectally, for example, as a suppository; parenterally by injection; or continuously by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion. A xe2x80x9ctherapeutically effective amountxe2x80x9d is intended to mean the amount of an agent that, when administered to a mammal in need thereof, is sufficient to effect treatment for injury or disease condition mediated by inhibition of PARP activity, such as for potentiation of anti-cancer therapies and inhibition of neurotoxicity consequent to stroke, head trauma, and neurodegenerative diseases. The amount of a given compound of the invention that will be therapeutically effective will vary depending upon factors such as the particular compound, the disease condition and the severity thereof, the identity of the mammal in need thereof, which amount may be routinely determined by artisans.
It will be appreciated that the actual dosages of the PARP-inhibiting agents used in the pharmaceutical compositions of this invention will be selected according to the particular complex being used, the particular composition formulated, the mode of administration and the particular site, and the host and condition being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage-determination tests. For oral administration, e.g., a dose that may be employed is from about 0.001 to about 1000 mg/kg body weight, with courses of treatment repeated at appropriate intervals.
Synthetic Processes
The present invention is further directed to methods of synthesizing the PARP-inhibiting agents by processes such as those set forth below for exemplary compounds of the invention. In the following examples, the structures of the compounds were confirmed by one or more of the following: proton magnetic resonance spectroscopy, infrared spectroscopy, elemental microanalysis, mass spectrometry, thin layer chromatography, high performance liquid chromatography, and melting point.
Proton magnetic resonance (1H NMR) spectra were determined using a 300 megahertz Tech-Mag, Bruker Avance 300DPX, or Bruker Avance 500 DRX spectrometer operating at a field strength of 300 or 500 megahertz (MHz). Chemical shifts are reported in parts per million (ppm, xcex4) downfield from an internal tetramethylsilane standard. Alternatively, 1 H NMR spectra were referenced to residual protic solvent signals as follows: CHCl3=7.26 ppm; DMSO=2.49 ppm; C6HD5=7.15 ppm. Peak multiplicities are designated as follows: s=singlet; d=doublet; dd=doublet of doublets; t=triplet; q=quartet; br=broad resonance; and m=multiplet. Coupling constants are given in Hertz (Hz). Infrared absorption (IR) spectra were obtained using a Perkin-Elmer 1600 series or a Midac Corporation FTIR spectrometer. Elemental microanalyses were performed by Atlantic Microlab Inc. (Norcross, Ga.) or Galbraith Laboratories (Nashville, Tenn.), and gave results for the elements stated within xc2x10.4% of the theoretical values. Flash column chromatography was performed using Silica gel 60 (Merck Art 9385). Analytical thin layer chromatography (TLC) was performed using precoated sheets of Silica 60 F254 (Merck Art 5719). Melting points (mp) were determined on a MelTemp apparatus and are uncorrected. All reactions were performed in septum-sealed flasks under a slight positive pressure of argon, unless otherwise noted. All commercial solvents were reagent-grade or better and used as supplied.
The following abbreviations may be used herein: Et2O (diethyl ether); DMF (N,N-dimethylformamide); DMSO (dimethylsulfoxide); MeOH (methanol); EtOH (ethanol); EtOAc (ethyl acetate); THF (tetrahydrofuran); Ac (acetyl); Me (methyl); Et (ethyl); and Ph (phenyl).
The general reaction protocols described below may be used to prepare the compounds of the invention. 
In Scheme 1,4-carbomethoxyindole A is formylated or acylated under various vilsmeier or Friedel-Crafts conditions to yield B, where R23 is CHO or COR24. 4-Carbomethoxyindole A serves as substrate for a 1,4-addition reaction to yield the nitroethyl intermediate B, where R23 is CHR25CH2NO2. Intermediate B, where R23 is CHO, is transformed to the corresponding oxime (R27 is CHxe2x95x90NOH) or nitroalkene (R27 is CHxe2x95x90CHNO2) C, which is then catalytically reduced to the aminoalkyl derivative D. Nitroethyl intermediate B is transformed directly to D (when R23 is CHR25CH2NO2) by reduction in some cases. Compound D spontaneously cyclizes to tricyclic lactams E (n=2) and EE. Exposure of intermediate D to basic conditions also leads to tricyclic lactams E and EE. Compound E is optionally N-alkylated to form N-alkylated E or halogenated to yield F. Intermediate F can be transformed via a metal-catalyzed reaction (typically with palladium as catalyst) into a number of different substituted tricyclic lactams G, where R29 is aryl, alkyl, alkenyl or alkynyl. G is optionally further modified at R22, R29 and R30.
Acyl-substituted compounds of formula J (e.g., compound 42) can be made by reaction with CO and the corresponding alcohol with Pd/C catalyst. The esters J may be further converted to other acyl derivatives by hydrolysis to the free acid, followed by activation to xe2x80x94C(O)-Lv, where Lv is a leaving group, by standard methods (e.g., March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th edition, August 1992, John Wiley and Sons, New York, ISBN 0471601802), and, for example, conversion to amides or other acyl derivatives by reactions generally known in the art. Alternatively, the esters J can be directly converted to amides by standard aminolysis reactions, e.g., by reaction with primary or secondary amines such as dimethylamine or pyrrolidine. 
R20=CO2CH3 
R21, R22=H
R23=COR24, (R24=H, aryl, (CH)qaryl), q=1 or 2
R29=optionally substituted aryl, alkyl, alkenyl, alkynyl,
R32=H, aryl, (CH2)qaryl)
cycloalkyl, heterocycloalkyl, or heteroaryl, or H.
In Scheme 2, intermediate BB, where R23 is CHO, (CO)aryl, or CO(CH2)qaryl where q is 1 or 2, is transformed to tricyclic acyl hydrazone H by reaction with hydrazine. 
In Scheme 3, the M, where Lv includes, for example, I, Br, or triflate, is coupled with a substituted alkyne T using palladium and copper catalysts (See e.g. Sonogashira, K., Tohda, Y., Ragihara, N. Tetrahedron Lett. 1975, 50, 4467-4470, incorporated herein by reference). The intermediate N can be cyclized with palladium catalyst (See e.g. Arcadi, A., Cacchu, S., Marinellito, F. Tetrahedron Lett. 1989, 30, 2581-2584, incorporated herein by reference) to give P which is further modified as described in Scheme 1 to the intermediate BB.